Osteocyte Biology Research Articles

The rise of osteocyte biology continues: Meeting report from the 32nd Annual Meeting of the American Society for Bone and Mineral Research

The rise of osteocyte biology continues: Meeting report from the 32nd Annual Meeting of the American Society for Bone and Mineral Research

The amazing osteocyte

The last decade has provided a virtual explosion of data on the molecular biology and function of osteocytes. Far from being the “passive placeholder in bone,” this cell has been found to have numerous functions, such as acting as an orchestrator of bone remodeling through regulation of both osteoclast and osteoblast activity and also functioning as an endocrine cell. The osteocyte is a source of soluble factors not only to target cells on the bone surface but also to target distant organs, such as kidney, muscle, and other tissues. This cell plays a role in both phosphate metabolism and calcium availability and can remodel its perilacunar matrix. Osteocytes compose 90% to 95% of all bone cells in adult bone and are the longest lived bone cell, up to decades within their mineralized environment. As we age, these cells die, leaving behind empty lacunae that frequently micropetrose. In aged bone such as osteonecrotic bone, empty lacunae are associated with reduced remodeling. Inflammatory factors such as tumor necrosis factor and glucocorticoids used to treat inflammatory disease induce osteocyte cell death, but by different mechanisms with potentially different outcomes. Therefore, healthy, viable osteocytes are necessary for proper functionality of bone and other organs. © 2011 American Society for Bone and Mineral Research.

Mechanosensing in Bone

The rationale for this issue of Clinical Reviews in Bone and Mineral Metabolism is rooted in the recognition of the revolution of the role of the osteocyte in bone biology. Since Wolff formulated his law of bone remodeling more than a century ago, the notion that mechanical function determines skeletal design and bone tissue organization has become widely accepted, and the mechanism whereby bone tissue detects mechanical loading is now becoming more and more clear. Basically, the cellular response mechanism to altered load starts with an adequate mechanical signal that is detected by the sensor cells, leading to the generation of an adequate signal that modulates bone formation and/or bone resorption. Bone formation and bone resorption result from the activity of osteoblasts and osteoclasts on the bone surface, while osteocytes are the cells within the tissue proper, and bone lining cells cover inactive bone surfaces. It is now known that osteocytes act as mechanosensors regulating mechanical adaptation and that load placed on an intact bone activates them. Our current knowledge of osteocytes still lags behind what we know of the properties and functions of both osteoblasts and osteoclasts, but the pace with which our knowledge of the osteocyte is advancing is impressive and illustrates an important role for osteocytes in bone biology. This issue of Clinical Reviews in Bone and Mineral Metabolism on ‘‘Mechanosensing in bone’’ is intended to review the concept and background information about the topic of mechanosensing in bone with the latest information on putative chemical/mechanical regulators along with speculation/impact on disease manifestations and is designed with a logic that might compel to read on. The first chapter, ‘‘Mechanisms of osteocyte mechanotransduction’’ by Bakker and Klein-Nulend, covers and provides a critical analysis of our current knowledge on how bone transduction takes place from the level of mechanical loads placed upon the bone as an organ to activation of a cell. The second chapter, ‘‘Application of bioimaging to osteocyte biology’’ by Kamioka and Adachi, is specifically devoted to the application of a new bioimaging technique to reveal the morphology of osteocytes as well as part of their biological nature. The application of this new technique has important relevance for understanding the osteocytes behavior in response to an extracellular stimulus. The third chapter, ‘‘Stress response by bone cells and implications on microgravity environment’’ by Bacabac and Van Loon, addresses the great advances that have been made in understanding the mechanisms involved in the adaptation of bone tissue to mechanical loading, leading to the notion that the mechanical properties of cells are a prerequisite to their tissue-level emergent mechanosensing behavior. A condition of disturbed mechanics due to extreme unloading, as in an environment under microgravity, may lead to impaired mechanosensing. The fourth chapter, ‘‘Gap junctions and biophysical regulation of bone cells’’ by Lloyd and Donahue, demonstrates the integral role of gap junctional intercellular communication in skeletal physiology and bone cell mechanosensing. This direct cell–cell communication via gap junctions is important for the communication between osteoblasts, osteoclasts, and osteocytes and is integral to J. Klein-Nulend (&) Department of Oral Cell Biology, Academic Centre for Dentistry, Amsterdam-University of Amsterdam and VU University Amsterdam, Research Institute MOVE, Gustav Mahlerlaan 3004, 1081 LA Amsterdam, The Netherlands e-mail: j.kleinnulend@acta.nl

Application of Bioimaging to Osteocyte Biology

In a variety of scientific fields, it is helpful to visualize natural phenomena. Newly developed methods of visualization often lead to breakthroughs in scientific fields. Especially, in the bioscience field, it is significant to reveal temporal–spatial responses in cells while visualizing molecular phenomena. Such visualization may provide information to help understand cellular behavior in response to an extracellular stimulus. Although osteocytes are the most abundant cells in bone, it has been difficult to study their biological nature because they are embedded in hard bone tissue. So, the real 3D structure of osteocytes was not clarified until recently. On the other hand, a newly developed technique of visualization was recently introduced into bone cell biology. In this review, we introduce and review our application of a bioimaging technique to reveal the morphology of osteocytes as well as part of their biological nature.

Sclerostin: A gem from the genome leads to bone-building antibodies

Sclerostin: A gem from the genome leads to bone-building antibodies

Human Primary Osteocyte Differentiation in a 3D Culture System

Studies on primary osteocytes, which compose >90-95% of bone cells, embedded throughout the mineralized matrix, are a major challenge because of their difficult accessibility and the very rare models available in vitro. We engineered a 3D culture method of primary human osteoblast differentiation into osteocytes. These 3D-differentiated osteocytes were compared with 2D-cultured cells and with human microdissected cortical osteocytes obtained from bone cryosections. Human primary osteoblasts were seeded either within the interspace of calibrated biphasic calcium phosphate particles or on plastic culture dishes and cultured for 4 wk in the absence of differentiation factors. Osteocyte differentiation was assessed by histological and immunohistological analysis after paraffin embedding of culture after various times, as well as by quantitative RT-PCR analysis of a panel of osteoblast and osteocyte markers after nucleic acid extraction. Histological analysis showed, after only 1 wk, the presence of an osteoid matrix including many lacunae in which the cells were individually embedded, exhibiting characteristics of osteocyte-like cells. Real-time PCR expression of a set of bone-related genes confirmed their osteocyte phenotype. Comparison with plastic-cultured cells and mature osteocytes microdissected from human cortical bone allowed to assess their maturation stage as osteoid-osteocytes. This model of primary osteocyte differentiation is a new tool to gain insights into the biology of osteocytes. It should be a suitable method to study the osteoblast-osteocyte differentiation pathway, the osteocyte interaction with the other bone cells, and orchestration of bone remodeling transmitted by mechanical loading and shear stress. It should be used in important cancer research areas such as the cross-talk of osteocytes with tumor cells in bone metastasis, because it has been recently shown that gene expression in osteocytes is strongly affected by cancer cells of different origin. It could also be a very efficient tool for drug testing and bone tissue engineering applications.

A systems biology approach to the identification and analysis of transcriptional regulatory networks in osteocytes

BackgroundThe osteocyte is a type of cell that appears to be one of the key endocrine regulators of bone metabolism and a key responder to initiate bone formation and remodeling. Identifying the regulatory networks in osteocytes may lead to new therapies for osteoporosis and loss of bone.ResultsUsing microarray, we identified 269 genes over-expressed in osteocyte, many of which have known functions in bone and muscle differentiation and contractility. We determined the evolutionarily conserved and enriched TF binding sites in the 5 kb promoter regions of these genes. Using this data, a transcriptional regulatory network was constructed and subsequently partitioned to identify cis-regulatory modules.ConclusionOur results show that many osteocyte-specific genes, including two well-known osteocyte markers DMP1 and Sost, have highly conserved clustering of muscle-related cis-regulatory modules, thus supporting the concept that a muscle-related gene network is important in osteocyte biology and may play a role in contractility and dynamic movements of the osteocyte.

Identification of differentially expressed genes between osteoblasts and osteocytes

Osteocytes represent the most abundant cellular component of mammalian bones with important functions in bone mass maintenance and remodeling. To elucidate the differential gene expression between osteoblasts and osteocytes we completed a comprehensive analysis of their gene profiles. Selective identification of these two mature populations was achieved by utilization of visual markers of bone lineage cells. We have utilized dual GFP reporter mice in which osteocytes are expressing GFP (topaz) directed by the DMP1 promoter, while osteoblasts are identified by expression of GFP (cyan) driven by 2.3 kb of the Col1a1 promoter. Histological analysis of 7-day-old neonatal calvaria confirmed the expression pattern of DMP1GFP in osteocytes and Col2.3 in osteoblasts and osteocytes. To isolate distinct populations of cells we utilized fluorescent activated cell sorting (FACS). Cell suspensions were subjected to RNA extraction, in vitro transcription and labeling of cDNA and gene expression was analyzed using the Illumina WG-6v1 BeadChip. Following normalization of raw data from four biological replicates, 3444 genes were called present in all three sorted cell populations: GFP negative, Col2.3cyan + (osteoblasts), and DMP1topaz + (preosteocytes and osteocytes). We present the genes that showed in excess of a 2-fold change for gene expression between DMP1topaz + and Col2.3cyan + cells. The selected genes were classified and grouped according to their associated gene ontology terms. Genes clustered to osteogenesis and skeletal development such as Bmp4, Bmp8a, Dmp1, Enpp1, Phex and Ank were highly expressed in DMP1topaz + cells. Most of the genes encoding extracellular matrix components and secreted proteins had lower expression in DMP1topaz + cells, while most of the genes encoding plasma membrane proteins were increased. Interestingly a large number of genes associated with muscle development and function and with neuronal phenotype were increased in DMP1topaz + cells, indicating some new aspects of osteocyte biology. Although a large number of genes differentially expressed in DMP1topaz + and Col2.3cyan + cells in our study have already been assigned to bone development and physiology, for most of them we still lack any substantial data. Therefore, isolation of osteocyte and osteoblast cell populations and their subsequent microarray analysis allowed us to identify a number or genes and pathways with potential roles in regulation of bone mass.

The biology of osteocytes

Osteocytes, the most abundant cell type in bone, remain the least characterized. Several theories have been proposed regarding their function, including osteolysis, sensing the strains produced in response to mechanical loading of bones, and producing signals that affect the function of osteoblasts and osteoclasts and hence, bone turnover. This review also discusses the role of osteocyte apoptosis in targeted bone remodeling and proposes that the occurrence of osteocyte apoptosis is consistent with the description of apoptosis as an essential homeostatic mechanism for the healthy maintenance of tissues.

Isolated primary osteocytes express functional gap junctions in vitro

The osteocyte is the most abundant cell type in bone and is embedded in mineralized bone matrix. Osteocytes are still poorly characterized because of their location and the lack of primary osteocyte isolation methods. Data on the cell biology of osteocytes is especially limited. We have isolated primary osteocytes from rat cortical bone by applying repeated enzymatic digestion and decalcification. The isolated osteocytes expressed typical osteocytic morphology with cell-cell contacts via long protrusions after a 1-day culture. These cells were negative or faintly positive for alkaline phosphatase but expressed high levels of osteocalcin, PHEX (phosphate-regulating gene with homology to endopeptidases on the X chromosome), and DMP1 (dentin matrix protein 1). These cells also revealed patchy membrane staining for connexin43. For studying the function of gap junctions in isolated osteocytes, we microinjected rhodamine-labeled dextran (MW: 10,000) and Lucifer yellow (MW: 457) and found that Lucifer yellow was rapidly transmitted to several surrounding cells, whereas dextran remained in the injected cells. Heptanol and 18alpha-glycyrrhetinic acid inhibited the transfer of Lucifer yellow. This clearly showed the existence of functional gap junctions in cultured osteocytes. Enveloped viruses, such as vesicular stomatitis virus and influenza A virus, were used for studying cell polarity. We were unable to demonstrate plasma membrane polarization with enveloped viruses in isolated primary osteocytes in culture. Our results suggest that osteocytes do not possess apical and basolateral plasma membrane domains as do osteoblasts, which are their precursors.

Osteocyte biology

Osteocyte biology

The osteocyte

Osteocytes are the most numerous cells in mature bone and have the potential to live as long as the organism itself. However, study and subsequent understanding of osteocyte biology has been thwarted by the remote location of the cell in the mineralized matrix. This review is intended to synthesize current understanding of osteocyte biology and to suggest future paths that will promote understanding of this obscure cell and translation of knowledge to disease prophylaxis and management. Cell facts • most abundant cells in mature bone; • average half-life of 25 years; • do not divide; • periosteocytic space provides largest surface area for ion exchange and molecular filtration in body.